The modulation of hydrolytic activity of penicillin V acylase (EC 3.5.1.11) from Streptomyces lavendulae by organic solvents is reported. On one hand, the addition of water-soluble cosolvents increases the catalytic activity up to a critical concentration of the non-aqueous solvents, yet further increase of the latter leads to protein denaturation. For alcohols and aprotic polar solvents, there are linear correlations between the critical concentration of water miscible cosolvent (at which enzyme deactivation does not begin to take place) and the dielectric constant of the cosolvents added. On the other hand, water-immiscible solvents can show activating or inhibitory effects that may be related to interactions between the structure of the solvent and the enzyme.